Comparison of Fabrication Methods Based on Nanoimprinting Lithography for Plasmonic Color Filter Fabrication

The angle-variable tunable optical filter was strictly fabricated by two strategies of nanoimprint-coupled metal nanopatterning with improved cost-effectiveness and accessibility. The tunable optical properties and the performances of two strategies were experimentally examined and turned out to be well matched to numerical results. Tunable properties are obtained by three factors: size of fabricated Ag nanodisks, incident illumination angle, and fabrication strategies. The resonant extinction peak shifts were identified to show a large increase along with the increase in fabricated Ag disk size and increase in the incidence angle of illumination. When comparing a fabrication strategy, it was confirmed that the sample fabricated by the strip-off method has better stability on color changes with a consistent dependency on the incident angle. The presented strategies of fabrication are technically viable for obtaining well-defined plasmonic nanostructures so that it has the feasibility to apply for fascinating optical applications including display or tunable optical filters.

     

An autism-associated variant of Epac2 reveals a role for Ras/Epac2 signaling in controlling basal dendrite maintenance in mice

The architecture of dendritic arbors determines circuit connectivity, receptive fields, and computational properties of neurons, and dendritic structure is impaired in several psychiatric disorders. While apical and basal dendritic compartments of pyramidal neurons are functionally specialized and differentially regulated, little is known about mechanisms that selectively maintain basal dendrites. Here we identified a role for the Ras/Epac2 pathway in maintaining basal dendrite complexity of cortical neurons. Epac2 is a guanine nucleotide exchange factor (GEF) for the Ras-like small GTPase Rap, and it is highly enriched in the adult mouse brain. We found that in vivo Epac2 knockdown in layer 2/3 cortical neurons via in utero electroporation reduced basal dendritic architecture, and that Epac2 knockdown in mature cortical neurons in vitro mimicked this effect. Overexpression of an Epac2 rare coding variant, found in human subjects diagnosed with autism, also impaired basal dendritic morphology. This mutation disrupted Epac2's interaction with Ras, and inhibition of Ras selectively interfered with basal dendrite maintenance. Finally, we observed that components of the Ras/Epac2/Rap pathway exhibited differential abundance in the basal versus apical dendritic compartments. These findings define a role for Epac2 in enabling crosstalk between Ras and Rap signaling in maintaining basal dendrite complexity, and exemplify how rare coding variants, in addition to their disease relevance, can provide insight into cellular mechanisms relevant for brain connectivity.     

Olfactory Marker Protein Expression Is an Indicator of Olfactory Receptor-Associated Events in Non-Olfactory Tissues

Olfactory receptor (OR)-associated events are mediated by well-conserved components in the olfactory epithelium, including olfactory G-protein (G_olf), adenylate cyclase III (ACIII), and olfactory marker protein (OMP). The expression of ORs has recently been observed in non-olfactory tissues where they are involved in monitoring extracellular chemical cues. The large number of OR genes and their sequence similarities illustrate the need to find an effective and simple way to detect non-olfactory OR-associated events. In addition, expression profiles and physiological functions of ORs in non-olfactory tissues are largely unknown. To overcome limitations associated with using OR as a target protein, this study used OMP with G_olf and ACIII as targets to screen for potential OR-mediated sensing systems in non-olfactory tissues. Here, we show using western blotting, real-time PCR, and single as well as double immunoassays that ORs and OR-associated proteins are co-expressed in diverse tissues. The results of immunohistochemical analyses showed OMP (+) cells in mouse heart and in the following cells using the corresponding marker proteins c-kit, keratin 14, calcitonin, and GFAP in mouse tissues: interstitial cells of Cajal of the bladder, medullary thymic epithelial cells of the thymus, parafollicular cells of the thyroid, and Leydig cells of the testis. The expression of ORs in OMP (+) tissues was analyzed using a refined microarray analysis and validated with RT-PCR and real-time PCR. Three ORs (olfr544, olfr558, and olfr1386) were expressed in the OMP (+) cells of the bladder and thyroid as shown using a co-immunostaining method. Together, these results suggest that OMP is involved in the OR-mediated signal transduction cascade with olfactory canonical signaling components between the nervous and endocrine systems. The results further demonstrate that OMP immunohistochemical analysis is a useful tool for identifying expression of ORs, suggesting OMP expression is an indicator of potential OR-mediated chemoreception in non-olfactory systems.     

MRPrimer: a MapReduce-based method for the thorough design of valid and ranked primers for PCR

Primer design is a fundamental technique that is widely used for polymerase chain reaction (PCR). Although many methods have been proposed for primer design, they require a great deal of manual effort to generate feasible and valid primers, including homology tests on off-target sequences using BLAST-like tools. That approach is inconvenient for many target sequences of quantitative PCR (qPCR) due to considering the same stringent and allele-invariant constraints. To address this issue, we propose an entirely new method called MRPrimer that can design all feasible and valid primer pairs existing in a DNA database at once, while simultaneously checking a multitude of filtering constraints and validating primer specificity. Furthermore, MRPrimer suggests the best primer pair for each target sequence, based on a ranking method. Through qPCR analysis using 343 primer pairs and the corresponding sequencing and comparative analyses, we showed that the primer pairs designed by MRPrimer are very stable and effective for qPCR. In addition, MRPrimer is computationally efficient and scalable and therefore useful for quickly constructing an entire collection of feasible and valid primers for frequently updated databases like RefSeq. Furthermore, we suggest that MRPrimer can be utilized conveniently for experiments requiring primer design, especially real-time qPCR.     

A cold-adapted tyrosinase with an abnormally high monophenolase/diphenolase activity ratio originating from the marine archaeon <Emphasis Type="Italic">Candidatus Nitrosopumilus koreensis</Emphasis>

Objectives

To obtain an acidic and cold-active tyrosinase, which potentially minimizes unwanted self-oxidation of tyrosinase-catalyzed catechols, including 3,4-dihydroxyphenylalanine at elevated pH and high temperature.

Results

A putative psychrophilic tyrosinase (named as tyrosinase-CNK) was identified from the genome information of the marine archaeon Candidatus Nitrosopumilus koreensis. This protein contains key tyrosinase domains, such as copper-binding domains and an O₂-binding motif, and phylogenetic analysis revealed that it was distinct from other known bacterial tyrosinases. Functional tyrosinase-CNK was produced by applying a co-expression strategy together with chaperone proteins in Escherichia coli with a yield of approx. 30 mg l^?1 and a purity >95 %. The purified enzyme showed optimal activity at pH 6 and 20 °C and still had 50 % activity at 0 °C. Surprisingly, the enzyme exhibited an abnormally high monophenolase/diphenolase activity ratio.

Conclusions

The acidic and cold-adapted tyrosinase-CNK, as a new type of tyrosinase, could expand potential applications of tyrosinases including the production of catechols through minimizing unwanted self-oxidation and the modification of existing materials at low temperature.

     

Generation of disease-specific induced pluripotent stem cells from patients with rheumatoid arthritis and osteoarthritis

Introduction

Since the concept of reprogramming mature somatic cells to generate induced pluripotent stem cells (iPSCs) was demonstrated in 2006, iPSCs have become a potential substitute for embryonic stem cells (ESCs) given their pluripotency and “stemness” characteristics, which resemble those of ESCs. We investigated to reprogram fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) to generate iPSCs using a 4-in-1 lentiviral vector system.

Methods

A 4-in-1 lentiviral vector containing Oct4, Sox2, Klf4, and c-Myc was transduced into RA and OA FLSs isolated from the synovia of two RA patients and two OA patients. Immunohistochemical staining and real-time PCR studies were performed to demonstrate the pluripotency of iPSCs. Chromosomal abnormalities were determined based on the karyotype. SCID-beige mice were injected with iPSCs and sacrificed to test for teratoma formation.

Results

After 14 days of transduction using the 4-in-1 lentiviral vector, RA FLSs and OA FLSs were transformed into spherical shapes that resembled embryonic stem cell colonies. Colonies were picked and cultivated on matrigel plates to produce iPSC lines. Real-time PCR of RA and OA iPSCs detected positive markers of pluripotency. Immunohistochemical staining tests with Nanog, Oct4, Sox2, Tra-1-80, Tra-1-60, and SSEA-4 were also positive. Teratomas that comprised three compartments of ectoderm, mesoderm, and endoderm were formed at the injection sites of iPSCs. Established iPSCs were shown to be compatible by karyotyping. Finally, we confirmed that the patient-derived iPSCs were able to differentiate into osteoblast, which was shown by an osteoimage mineralization assay.

Conclusion

FLSs derived from RA and OA could be cell resources for iPSC reprogramming. Disease- and patient-specific iPSCs have the potential to be applied in clinical settings as source materials for molecular diagnosis and regenerative therapy.